This project is a continuation of a project initiated in 1996 (see Highlight #91). Ribonucleotide reductases (RNRs) catalyze the first step of the DNA biosynthesis: the conversion of nucleotides to deoxynucleotides. RNR consists of two units: a large unit (R1) and a R2 unit which is small(er). The latter unit contains a tyrosyl radical. This radical initiates the nucleotide reduction by means of a long range coupled electron proton transfer reaction. When R2 is treated with iron, a di-ferrous complex is generated, which rapidly is transformed into a di-ferric center while a stable tyrosyl radical is being formed. This transformation involves a step in which the di-ferrous complex is changed into an intermediate (called Intermediate X). As has been shown by resonant Raman experiments, the di-iron center in R2 contains an oxygen bridge between the two irons. It is of interest to learn if the oxygen bridge in Intermediate X consists of one or two oxygen atoms. The answer to this question may reveal insight in the chemical reactions involved in the formation of the tyrosyl radical. In an attempt to elucidate the problem, the following experiments have been performed. S-band CW EPR spectra have been taken of both an unlabeled and a 17O labeled-sample of Intermediate X. We expect the EPR line of the labeled sample to have a different shape and width than that of the unlabeled sample due to the hyperfine broadening. Spectra obtained in 1996 could be simulated but the best simulations used 75% isotopic oxgyen, 17O, while 85% enrichment was expected. In the spring of 1997, low-frequency S-band spectra were obtained on new samples and a new loop-gap resonator. Better signal to noise was obtained and the spectra could be simulated using 85% 17O. Apparently either the sample or the resonator (or both) had less radical signal which was not considered in the simulations. It is concluded that Intermediate X consists of a bridge from one 17O atom. [unreadable]